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Circadian clocks (oscillators) regulate multiple aspects of insect behaviour and physiology. The circadian system located in the male reproductive tract of Lepidoptera orchestrates rhythmic sperm release from testis and sperm maturation in the upper vas deferens (UVD). Our previous research on the cotton leafworm, Spodoptera littoralis, suggested rhythmic changes in the V-ATPase levels in the UVD epithelium, which correlated with rhythmic pH fluctuations in the UVD lumen. However, it was not known whether UVD cells contain clock mechanism that generates these daily fluctuations. In the current paper, we show circadian rhythm in the expression of clock gene period at the mRNA and protein level in the UVD epithelium. To determine the role of PER in V-ATPase and pH regulation, testes–UVD complexes were treated in vitro with double-stranded fragments of per mRNA (dsRNA). This treatment, which transiently lowered per mRNA and protein in the UVD, altered expression of V-ATPase c subunit. In addition, per RNAi caused a significant delay in the UVD lumen acidification. These data demonstrate that the UVD molecular oscillator involving the period gene plays an essential role in the regulation of rhythmic V-ATPase activity and periodic acidification of the UVD lumen.

Disruption of the circadian clock, which directs rhythmic expression of numerous output genes, accelerates aging. To enquire how the circadian system protects aging organisms, here we compare circadian transcriptomes in heads of young and old Drosophila melanogaster. The core clock and most output genes remained robustly rhythmic in old flies, while others lost rhythmicity with age, resulting in constitutive over- or under-expression. Unexpectedly, we identify a subset of genes that adopted increased or de novo rhythmicity during aging, enriched for stress-response functions. These genes, termed late-life cyclers, were also rhythmically induced in young flies by constant exposure to exogenous oxidative stress, and this upregulation is CLOCK-dependent. We also identify age-onset rhythmicity in several putative primary piRNA transcripts overlapping antisense transposons. Our results suggest that, as organisms age, the circadian system shifts greater regulatory priority to the mitigation of accumulating cellular stress., Disruption of circadian rhythms leads to reduced healthspan, but the mechanisms by which the normal clock protects aging organisms are not known. Here, the authors show that a subset of genes becomes more rhythmically expressed in older flies, and these are enriched for response to oxidative stress.

Bacterial small noncoding RNAs (sRNAs) have been discovered in many genetically well-studied microorganisms and have been shown to regulate critical cellular processes at the post-transcriptional level. In this study, we used comparative genomics and microarray data to analyze the genome of the ammonia-oxidizing bacterium Nitrosomonas europaea for the presence and expression of sRNAs. Fifteen genes encoding putative sRNAs (psRNAs) were identified. Most of these genes showed altered expression in a variety of experimental conditions. The transcripts of two psRNAs were further characterized by mapping their 5'- and 3'-ends and by real-time PCR. The results of these analyses suggested that one of them, psRNA11, is involved in iron homeostasis in N. europaea.

The process of nitrification has the potential for the in situ bioremediation of halogenated compounds provided a number of challenges can be overcome. In nitrification, the microbial process where ammonia is oxidized to nitrate, ammonia-oxidizing bacteria (AOB) are key players and are capable of carrying out the biodegradation of recalcitrant halogenated compounds. Through industrial uses, halogenated compounds often find their way into wastewater, contaminating the environment and bodies of water that supply drinking water. In the reclamation of wastewater, halogenated compounds can be degraded by AOB but can also be detrimental to the process of nitrification. This minireview considers the ability of AOB to carry out cometabolism of halogenated compounds and the consequent inhibition of nitrification. Possible cometabolism monitoring methods that were derived from current information about AOB genomes are also discussed. AOB expression microarrays have detected mRNA of genes that are expressed at higher levels during stress and are deemed "sentinel" genes. Promoters of selected "sentinel" genes have been cloned and used to drive the expression of gene-reporter constructs. The latter are being tested as early warning biosensors of cometabolism-induced damage in Nitrosomonas europaea with promising results. These and other biosensors may help to preserve the tenuous balance that exists when nitrification occurs in waste streams containing alternative AOB substrates such as halogenated hydrocarbons.

Transcriptional fusions with gfp driven by the promoter region of mbla (NE2571) in pPRO/mbla4 and clpB (NE2402) in pPRO/clpb7 were used to transform the ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718). The two genes were chosen because their transcript levels were found at much higher levels in N. europaea in response to oxidation of chloroform and chloromethane. In N. europaea transformed with pPRO/mbla4, green fluorescent protein (GFP)-dependent fluorescence increased from 3- to 18-fold above control levels in response to increasing chloroform concentrations (7 to 28 microM), and from 8- to 10-fold in response to increasing hydrogen peroxide concentrations (2.5-7.5 mM). The GFP-dependent fluorescence of N. europaea transformed with pPRO/clpb7 also showed an increase of 6- to 10-fold in response to chloroform (28-100 microM) but did not respond to H(2)O(2). Our data provide proof of concept that biosensors can be fabricated in ammonia-oxidizing bacteria using "sentinel" genes that up-regulate in response to stress caused either by co-oxidation of chlorinated solvents or by the presence of H(2)O(2). The fabricated biosensors had a consistent concentration-dependent response to chloroform; however, these did not respond to other chlorinated compounds that cause similar cellular stress.

Circadian clock genes are ubiquitously expressed in the nervous system and peripheral tissues of complex animals. While clock genes in the brain are essential for behavioral rhythms, the physiological roles of these genes in the periphery are not well understood. Constitutive expression of the clock gene period was reported in the ovaries of Drosophila melanogaster; however, its molecular interactions and functional significance remained unknown. This study demonstrates that period( per) and timeless( tim) are involved in a novel noncircadian function in the ovary. PER and TIM are constantly expressed in the follicle cells enveloping young oocytes. Genetic evidence suggests that PER and TIM interact in these cells, yet they do not translocate to the nucleus. The levels of TIM and PER in the ovary are affected neither by light nor by the lack of clock-positive elements Clock( Clk) and cycle( cyc). Taken together, these data suggest that per and tim are regulated differently in follicle cells than in clock cells. Experimental evidence suggests that a novel fitness-related phenotype may be linked to noncircadian expression of clock genes in the ovaries. Mated females lacking either per or tim show nearly a 50% decline in progeny, and virgin females show a similar decline in the production of mature oocytes. Disruption of circadian mechanism by either the depletion of TIM via constant light treatment or continuous expression of PER via GAL4/UAS expression system has no adverse effect on the production of mature oocytes.

Release of sperm bundles from moth testes is controlled by the local circadian oscillator. The mechanism which restricts migration of sperm bundles to a few hours each day is not understood. We demonstrate that a daily cycle of sperm release is initiated by the migration of folded apyrene sperm bundles through a cellular barrier at the testis base. These bundles have conspicuous concentrations of actin filaments at their proximal end. Inhibition of actin polymerization by cytochalasin at a specific time of day inhibited sperm release from the testis. Likewise, application of double-stranded actin RNA specifically inhibited sperm release. This RNA-mediated interference (RNAi) lowered the pool of actin mRNA in tissues involved in sperm release. The decline in mRNA levels resulted in the selective depletion of F-actin from the tip of apyrene sperm bundles, suggesting that this actin may be involved in the initiation of sperm release. Combined results of RNAi experiments at physiological, cellular and molecular levels identified unique cells that are critically involved in the mechanism of sperm release.

The authors examined patterns of spatial and temporal expression of Drosophila per gene homologue in the codling moth, Cydia pomonella. Since sperm release in moths is regulated in a circadian manner by an autonomous clock that is independent from the brain, the authors investigated per expression in male reproductive system along with its expression in moth heads. per mRNA is rhythmically expressed with the same phase and amplitude in both tissues under light-dark (LD) conditions. The levels of per mRNA are low during the day, start to increase before lights-off, reach the peak in dark, and decrease after lights-on. In constant darkness (DD), cycling of per mRNA continued in heads with severely blunted amplitude. No cycling of per mRNA was detected in testis in DD. In situ hybridization and immunocytochemistry revealed distinct spatial patterns of per expression in the moth reproductive system. There is no expression of per in cells forming the wall of testes or in sperm bundles. However, permRNA and protein are rhythmically expressed in the epithelial cells forming the wall of the upper vas deferens (UVD) and in the cells of the terminal epithelium, which are involved in the circadian gating of sperm release. Increase in permRNA in the UVD coincides with sperm accumulation in this part of the insect reproductive system.

Thioredoxins and glutaredoxins are small ubiquitous redox proteins that were discovered as hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis. Some organisms encode more than one redox protein. In this study, we demonstrate that an open reading frame in the bacteriophage T4 genome, reported earlier and designated as Y55.7 (Tomaschewski, J., and Rüger, W. (1987) Nucleic Acids Res. 15, 3632-3633), encodes a second functional redox protein. Gene y55.7 was cloned and expressed in Escherichia coli. Purified Y55.7 protein had glutathione-dependent thioltransferase and dehydroascorbate reductase activities indicative of a functional glutaredoxin. The protein is expressed at all stages of the T4 infection cycle and can serve as a hydrogen donor for the phage ribonucleotide reductase in in vitro experiments.

Bacterial small noncoding RNAs (sRNAs) have been discovered in many genetically well-studied microorganisms and have been shown to regulate critical cellular processes at the post-transcriptional level. In this study, we used comparative genomics and microarray data to analyze the genome of the ammonia-oxidizing bacterium Nitrosomonas europaea for the presence and expression of sRNAs. Fifteen genes encoding putative sRNAs (psRNAs) were identified. Most of these genes showed altered expression in a variety of experimental conditions. The transcripts of two psRNAs were further characterized by mapping their 5'- and 3'-ends and by real-time PCR. The results of these analyses suggested that one of them, psRNA11, is involved in iron homeostasis in N. europaea.

Upon exposure of Nitrosomonas europaea to chloroform (7 μM, 1 h), transcripts for 175 of 2,460 genes were found at higher levels in treated cells than in untreated cells and transcripts for 501 genes were found at lower levels. With chloromethane (3.2 mM, 1 h), transcripts for 67 genes were at higher levels and transcripts for 148 genes were at lower levels. Transcripts for 37 genes were at higher levels following both treatments and included genes for heat shock proteins, σ-factors of the extracytoplasmic function subfamily, and toxin-antitoxin loci. N. europaea has higher levels of transcripts for a variety of defense genes when exposed to chloroform or chloromethane.

The period (per) and timeless (tim) genes encode key components of the circadian oscillator in Drosophila melanogaster. The per gene is thought to encode three transcripts via differential splicing (types A, B, and C) that give rise to three proteins. Since the three per mRNA types were based on the analysis of cDNA clones, we tested whether these mRNA types were present in vivo by RNase protection assays and reverse transcriptasemediated PCR. The results show that per generates two transcript types that differ only by the presence (type A) or absence (type B*) of an alternative intron in the 3* untranslated region. Transgenic flies containing transgenes that produce only type B* transcripts (per B* ), type A transcripts (per A ), or both transcripts (per G ) rescue locomotor activity rhythms with average periods of 24.7, 25.4, and 24.4 h, respectively. Although no appreciable differences in type A and type B* mRNA cycling were observed, a slower accumulation of PER in flies making only type A transcripts suggests that the intron affects the translation of per mRNA. The period (per) and timeless (tim) genes encode key components of the circadian oscillator in Drosophila melanogaster. The expression of these genes is required for circadian clock function, and an important aspect of their expression is circadian fluctuations in their mRNA and protein levels (17). These rhythms in per and tim gene products are controlled by a circadian feedback loop in which PER and TIM proteins control the expression of their own mRNAs (17, 25, 26). This feedback is mediated predominantly at the transcriptional level, though posttranscriptional regulation is also involved (5, 16, 29, 31, 32). The role of PER in this process is unknown, but its lack of a known DNA binding domain and inability to bind DNA indicate that it does not regulate transcription directly (17, 25). Analysis of per cDNA clones uncovered three splice variants that encode three different PER isoforms (3). The most abundant of these transcripts, type A, defined both the structure of the per gene and the prototypical 1,218-amino-acid PER protein. Type B transcripts differ from type A by having two additional introns; one removes 288 nucleotides (nt) from exon 5 of type A transcripts, and the other excises 89 nt from the 39 untranslated region (39UTR) of exon 8. After excising the intron from exon 5, type B transcripts produce a protein that is 96 amino acids shorter. The least abundant transcript (only one partial cDNA clone was isolated), type C, differs from type A by retaining introns 5, 6, and 7, thereby producing a transcript whose exon 5 spans exons 5 to 8 in type A transcripts. Due to the inclusion of these additional introns, the last 107 amino acids of the putative type C protein sequence are entirely different from the last 149 amino acids of type A protein sequence. All three per cDNAs are capable of rescuing behavioral rhythms in per 01 flies, though the type C construct may mediate behavioral rescue by generating both type A and type B transcripts (3, 4). Given the critical role that PER plays in controlling the circadian feedback loop in Drosophila, it is important to determine which isoforms contribute to the feedback loop mechanism and what impact this contribution may have on behavioral rhythms. Since the initial characterization of per mRNA splice variants was based on the structure and abundance of partial cDNA clones, we tested whether these per transcripts exist in vivo and function equally to rescue locomotor activity rhythms. Our studies failed to detect per splice variants that generate different PER isoforms. However, two per transcripts that differ by an alternatively spliced intron within their 39UTRs were found; type A contains the 89-bp intron, and type B9 lacks this intron. Transgenes that produce type A mRNA, type B9 mRNA, or both mRNA types each rescue robust locomotor activity rhythms, but the period of these rhythms tends to be longer in the transgene that produces only type A transcripts. Type A and type B9 transcripts are indistinguishable with respect to circadian cycling, but the levels of PER derived from the transgene expressing only type A transcripts rise with a later phase than PER derived from transgenes expressing only type B9 or both type A and type B9 transcripts. These results suggest that the alternatively spliced intron alters the translation of per mRNA.

Background Reproductive systems of male moths contain circadian clocks, which time the release of sperm bundles from the testis to the upper vas deferens (UVD) and their subsequent transfer from the UVD to the seminal vesicles. Sperm bundles are released from the testis in the evening and are retained in the vas deferens lumen overnight before being transferred to the seminal vesicles. The biological significance of periodic sperm retention in the UVD lumen is not understood. In this study we asked whether there are circadian rhythms in the UVD that are correlated with sperm retention.